Project Summary Dilated cardiomyopathy caused by mutations in the lamin A/C gene (LMNA) encoding A-type nuclear lamins is a life-threatening disease with no definitive cure. The pathogenic mechanisms responsible for cardiomyopathy in this inherited disease are poorly understood. In particular, it is not known how alterations in proteins expressed in nuclei of virtually all terminally differentiated cells selectively cause heart disease. Our hypothesis is that alterations in A-type lamins predispose cells to oxidative stress-induced remodeling of ryanodine receptors (RyRs), creating a sarcoplasmic reticulum (SR) Ca2+ ?leak.? Oxidative stress and increased cytosolic Ca2+ also contribute to hyper-activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), which occurs in cardiomyopathy caused by LMNA mutations. The increased cytosolic Ca2+ and ERK1/2 activity generates various defects, including mitochondrial dysfunction, that cause cardiomyopathy. A corollary of our hypothesis is that blocking the SR Ca2+ ?leak? will have beneficial effects in cardiomyopathy caused by LMNA mutations. Using mouse models of the disease and human tissue, we will test our hypothesis and its corollary. In Aim 1, we will determine if alterations in A-type lamins that cause cardiomyopathy lead to enhanced cardiac muscle oxidative stress, resultant RyR2 remodeling and a SR Ca2+ ?leak.? We will also determine if the SR Ca2+ ?leak? stimulates ERK1/2 activity, causes mitochondrial dysfunction and damages DNA. In addition to heart, we will similarly examine skeletal muscle, which is often simultaneously affected in human patients with cardiomyopathy caused by LMNA mutations as well as in model mice. We will further assess these processes in cultured cells that stably express a cardiomyopathy-causing lamin A variant or lack A-type lamins. In Aim 2, we will utilize the three-dimensional structure of RyR to determine how specific oxidative modifications that occur in striated muscle of Lmna mutant mice affect its structure and make it ?leaky? to Ca2+. In Aim 3, we will perform experiments to determine if a Rycal, drugs that stabilize remodeled RyRs and block the SR Ca2+ ?leak,? improves heart function and prolongs survival in Lmna mutant mice and if it blocks the ?leak? in hearts from human subjects with cardiomyopathy caused by LMNA mutations. We will further determine if a Rycal has synergistically beneficial effects when combined with an inhibitor of ERK1/2 activity, which has previously been shown to partially improve heart function in Lmna mutant mice with cardiomyopathy. These studies will reveal basic information about the pathogenesis of cardiomyopathy caused by LMNA mutations and connect an intranuclear protein defect with a tangible mechanism of cardiac dysfunction. They will also determine if drugs already in clinical development can be translated to trials in patients with this lethal heart disease.